JPS63190806A - Controlling of soil bright of vegetables by plant root plant bacteria - Google Patents

Abstract

PURPOSE:To suppress soil blights of vegetables caused by fungi of the genus Pythium or Rhizoctonia, by applying and growing a microorganism of the family Pseuodomonadeceae by a method for coating seeds of the vegetables with the microorganism or dipping seedlings in a microbial culture fluid, etc. CONSTITUTION:Pseudomonas fluorescens SCB-8004, Pseudomonas fluorescens SCB-10705, Pseudomonas fluorescens SCB-10807, Xanthomonas sp. SCB-12801 and Pseudomonas sp. SCB-16701 are applied to and grown on vegetables to suppress soil blights, e.g. damping-off of cucurbitaceous vegetables, caused by fungi of the genus Pythium or Rhizoctonia and cultivate healthy vegetables. Methods for coating seeds of the vegetables with the microorganism, dipping seedlings in a microbial culture fluid, etc., are cited as the method for applying and growing the microorganism on the vegetables.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for cultivating healthy vegetables by suppressing soil diseases of vegetables using microorganisms, and a novel microorganism belonging to the family Pseutomonataceae used therein. More specifically, the present invention provides Pseudomonas florescens SCB-8004
Strain, Pseudomonas florescens SCB-1070
5 strains, Pseudomonas florescens SCB-10
807 strain, Xanthomonas sp. SCB-12801
strains, and Pseudomonas sp. A method for cultivating healthy vegetables by suppressing soil diseases caused by Pythium and Rhizoctonia by attaching and growing SCB-16701 strain on vegetables, and a novel microorganism, Pseudomonas sp., used in the above method. , SCB 167
01 strain, Xanthomonas sp, SCB 12801
Regarding stocks.

<Prior Art and Problems> In recent years, as vegetables have become a specialty and long-term continuous cultivation has progressed, the damage caused by soil diseases to vegetables has become serious. It has become clear that most of these soil diseases are caused by the abnormal proliferation of pathogenic bacteria in the soil, and their infection and decay. Therefore, in order to control this soil disease, many fungicides, soil fumigants, etc. have been developed and used. These pesticides cannot be sufficiently effective when sprayed or applied into the soil, and when applied in large quantities, they also kill beneficial microorganisms in the soil, resulting in a soil ecology suitable for growing crops. On the other hand, this may lead to the emergence of drug-resistant pathogens. There are concerns about the effects of other fumigants on workers. In order to compensate for these shortcomings of disease control using chemical pesticides, microorganisms that have antagonistic activity against disease-causing bacteria are isolated from the soil and added to plants directly or on field crops to prevent infection of crops with disease-causing bacteria and field crops. Biological control methods have been developed to suppress the growth or disease effects of pathogenic bacteria in soil. Although this method has a slow bactericidal effect, it does not disturb the soil ecology over a long period of time, suppresses the abnormal growth of pathogenic bacteria, and can suppress damage to crops caused by pathogenic bacteria.

Examples of technologies related to this matter are as follows.

An example of application to Pythium disease is the use of Trichoderma fungi for bean rot (Locke, J.C.;
pathology (1976) 69.536)
, an example of using Pseudomonas florescens or its antibacterial substance piolterin for cotton seedling damping-off (lowe
ll.

C,R,et,al, ; Phytopatholo
gy (1980) 70.712-715L An example of using rhizobial microorganisms to treat seedling blight of sugar beets (Osburn
+R,M,et,al, ;Phytopatholo
gy (1983) 1, 961). In addition, as an example of application of Trichoderma hamatum to daikon radish and beans as an example of application to diseases caused by Pythium bacterium or Trichoderma hamatum (Harman, G, E, et.

al,; Phytopatholog V (1980
) 70. 116”7-1172), an example of applying Gliocladium virens to cotton seedling blight (How
ellSC,R.

Phytopathology (1982) 72.4
96-498). Both research results demonstrate the usefulness of biological control, but when used in actual fields, there are problems such as the effectiveness being inconsistent depending on the variety of cultivated crops, land, climate conditions, etc. Furthermore, no examples have been shown to be effective against seedling damping-off of cucurbitaceous vegetables such as cucumbers.

<Means for Solving the Problem> In order to solve these problems, the present inventors aimed to (i) suppress the growth and infection of Pythium and Rhizoctonia bacteria from the root surfaces of healthy vegetables cultivated in continuous cultivation fields; (ii) It attaches to and grows on the rooting part from the seed, and (i
ii) Bacterial strains that grow vigorously on the root surface without dispersing into the field soil (iv) The microorganisms themselves do not cause disease to crops, etc. Strains are considered Bicillum bacteria, and Rhizoctonia bacteria are used as indicator disease bacteria. It was isolated by measuring the inhibitory activity of cucumber seedling damping-off disease.

Isolated bacteria. SCB-8004, SCB-10705
, SCB-10807, SCB-12801, and SC
The taxonomic properties of the five strains of B-16701 are shown in Table 1.

For each strain, purge aids manual op systematic bacteriology (Bergey's
Manual of Systematic Bact
Search results according to the search formula (Eriology). S.C.
B-8004 strain, SCB-10705 strain and SCB-
As shown in Table 1, all strains 10807 are Durham-negative rods and are motile aerobic bacteria, and they do not accumulate parahydroxybenzoate in their cells.
It was identified as Pseudomonas florescens based on its characteristics such as producing a fluorescent dye in King's B medium, having arginine dihydrolase activity, positive gelatin liquefaction ability, and not producing biocyanin.

The SCB-12801 strain is a motile aerobic Durham-negative bacillus that accumulates yellow pigment in its cells and produces mucoids on nutrient agar plates, making it similar to Xanthomonas campestris (Xanthomonas ca
mpestris) and Xanthomonas fragariae (
Xanthomonas fragariae) is mentioned as a similar bacterium. However, Xanthomonas campestris produces acid from fructose and trehalose. The SCB-12801 strain does not produce acid from them, and Xatomonas fragariae does not hydrolyze Escalin or degrade milk. The SCB-12801 strain differs from both of them in that it decomposes them by more than 0. S.C.
Strain B-12801 was recognized as a new bacterial species belonging to the genus Xanthomonas, and Xanthomonas sp. It was named SCB-12801.

The SCB-16701 strain is a Durham-negative bacillus, a motile aerobic bacterium, and no accumulation of bara-hydroxybenzoate is observed within the cells, and no fluorescent dye is produced in any culture medium. Pseudomonas stuteeri (Pseudomonas stuteeri), a member of the Pseudomonas genus, is
as 5 tutzeri) and Pseudomonas mendocina
are considered to be similar bacteria. However, although Pseudomonas stuteeri does not liquefy gelatin, it does
The 16701 strain is liquefied and also has the advantage of being a Pseudomonas strain.
Mendocina contains yellow or orange pigments within its cells and does not hydrolyze starch or liquefy gelatin. S.C.
B-16701 strain does not contain these pigments, and each of them is hydrolyzed,
It differs from both in that it liquefies. Therefore. SCB-167
Strain 01 is mycologically similar to but different from Pseudomonas stettieri and Pseudomonas mendocina. The SCB-16701 strain was recognized as a new bacterial species belonging to the genus Pseudomonas, and Pseudomonas sp, SCB 167
It was named 01.

Pseudomonas florescens SCB-80 above
04 strain, Pseudomonas florescens SCB-1
0705 strain, Pseudomonas florescens SCB
-10807 strain, Xanthomonas sp. SCB-12
801 strain and Pseudomonas sp, SC81670
One strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession numbers 9137, 9138, 9139, 9140, and 9141, respectively.

Isolation of these bacterial strains was performed by the following method.

Separate the healthy vegetables with sterilized scissors, shake off the attached soil in sterile water, take 5 fine roots cut to a length of about 1 CI11, put them in 5n+1 sterile water, and mix vigorously with a thermomixer for 5 minutes. The bacteria attached to the roots were released by stirring. The obtained solution was diluted 100 times and 10,000 times with sterilized water, 100 μl of the solution was spread on a nutrient agar medium (manufactured by Difco), and cultured at 27° C. for 2 to 4 days to form colonies.

The obtained colonies were spread on a nutrient agar medium and incubated at 27°C.
After culturing for 2 days, the grown bacterial cells were scraped off.

This method produces approximately 1,800 vegetables from 130 vegetable roots of 23 types.
of bacteria were obtained.

The bacteria thus isolated were cultured and applied to cucumber seeds whose surfaces had been sterilized in advance. The coated seeds were sown on a diseased surface containing Bicilium and Rhizoctonia, and the percentage of healthy seedlings was measured to obtain five particularly effective strains.

For culturing these strains, various media containing carbon sources, nitrogen sources, inorganic substances, etc. that are commonly used in the culture of microorganisms can be used. Specifically, carbon sources include glucose, fructose, maltose, galactose, ribose, sucrose, starch, starch powder hydrolyzate, sugars such as 11-density and waste F-densates, natural carbohydrates such as wheat and rice, glycerol, Use from common carbon sources such as alcohols such as mannitol, methanol, and ethanol, fatty acids such as gluconate, pyruvic acid, acetic acid, and citric acid, and amino acids such as glycine, glutamic acid, glutamine, alanine, and asparagine. One or more types may be appropriately selected and used in consideration of the assimilation ability of microorganisms.

Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein, gazamino acid, various amino acids, corn syrup liquor, fitschmeal or its hydrolyzate, and other animal,
Considering the assimilation ability of the microorganisms used from organic nitrogen compounds such as hydrolysates of plants and microorganisms, ammonium salts such as ammonia, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea. One kind or two or more kinds thereof may be appropriately selected and used.

In addition, trace amounts of magnesium, manganese,
One or more types of phosphates, hydrochlorides, sulfates, carbonates, acetates, etc. of iron, zinc, copper, sodium, calcium, potassium, etc. are added as appropriate, and if necessary, vegetable oil,
Antifoaming agents such as surfactants may also be added.

A solid medium prepared by adding a gelling agent such as agar to a medium containing a combination of these carbon sources, nitrogen sources, and inorganic salts can also be used.

Cultivation can be carried out using conventional culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture in a liquid medium containing the above-mentioned medium components, or a static culture method suitable for the microorganism used on a solid medium. Select and carry out the culture method.

The culture conditions may be selected as appropriate depending on the type of medium and culture method, and there are no particular restrictions as long as the strain can proliferate and soil diseases of vegetables caused by Pythium and Diphtonia can be controlled. , 25 to 33°C, and the pH of the culture solution is preferably about 6.5 to 8.

Five bacterial strains obtained as described above, Pseudomonas
Floreffense SCB-8004, Shade Monas
Floreffscens SCB-10705, Pseudomonas Floreffcens SCB-10807, Xanthomonas sp. SCB-12801, and Pseudomonas
sp. Vegetable soil diseases to which SCB-16701 is applied may be caused by Pythium or Rhizoctonia. Examples of diseases caused by Rhizoctonia fungi include seedling damping-off of tomatoes, eggplants, and other solanaceous vegetables, mg disease of strawberries, and diseases caused by Bicillum and Diphtonia fungi such as Ento, Tobacco, etc. It is effective against seedling damping-off of cotton, spinach, sugar beet, and dahlia. In addition, in implementing soil disease control of vegetables, methods for attaching and growing the five bacterial strains mentioned above on vegetables include, for example, a method of coating vegetable seeds, or a method of immersing vegetable seedlings in a bacterial culture solution. can be given.

Next, the present invention will be explained with reference to examples, but it goes without saying that the scope of the present invention is not limited in any way by these examples.

Example 1 Shademonas sp., SCB 16701, Xanthomonas sp. Add an equal amount of 2% of the five strains of SCB-12801, Pseudomonas floreffcens SCB-8004, Pseudomonas floreffcens SCB-10705, and Pseudomonas floreffcens SCB-10807 to a test bacterial solution cultured in King's B liquid medium at 27°C for 4 hours. Added methylcellulose. The solution 21 was added to 12 cucumber seeds whose surface had been previously sterilized with 1% antiformin and dried in a small container. V8
Pythium ultimum was inoculated into a wheat bran medium supplemented with Pythium ultimum, cultured at 27°C for 2 weeks, thoroughly dried, and crushed. Add and mix as shown, and add 110 ml to a 150 ml pot.

The number of bacteria attached to the seeds is 10' or more per seed. The coated seeds were sown in the pot soil, and 27
It was grown for 12 days in a °C greenhouse. The number of healthy bacteria without seedling damping-off was measured, and the disease suppression activity was tested.

(Composition of King's B medium) Glycerin 10ml Peptone
20g hydrogen phosphate dicarimum
1.5g magnesium sulfate heptahydrate 1.5g distilled water 1.0OOn+1 (pH 7,
2) (Bran medium with VB juice added) Bran medium 300I! Add 150ml of V8 Geniece (manufactured by Campbell) to ll, mix well, and heat to 121°.
C. Perform autoclaving for 20 minutes.

The results are shown in Table 2.

Table 2 Inhibitory activity against cucumber seedling damping-off (Bicillum 13III) Percentage of healthy seedlings (%) Skin damage concentration (g/I) SCB8004 100 88 69 50SCB
10705 100 92 78 65SCB 10
807 100 65 92 52SCB 1280
1 100 63 80 70SCB 16701
100 27, 53 75 No treatment 100 38
15 11 As shown in Table 2, when coated with the above bacteria, the percentage of healthy seedlings was higher than when untreated.
ultimum) was found to have the activity of suppressing seedling damping-off and disease.

Example 2 Rhizoctonia solani (Rhizoctonia 5) was also used as a pathogen for cucumber seedlings.
An experiment similar to that in Example 1 was conducted using .olani).

The results are shown in Table 3.

Table 3 Inhibitory activity against cucumber seedling damping-off (Rhizoctonia) SCB, 10705 100 46
47SCB 10807 100 4
7 50SCB 12801 100
11 19SCB 16701 100
33 38 No treatment 100 5 2 As in Example 1, when coated with the above bacteria, the percentage of healthy seedlings was higher than that without treatment.
Both of these bacteria are Rhizoctonia solani (Rh1zoc).
tonia 5olani) was found to have inhibitory activity against seedling damping-off and other diseases.

Claims (6)

[Claims] (1) Pseudomonas florescens SCB-80
04(￥Pseudomonas￥fluoresc
Pseudomonas florescens SCB-107
05(￥Pseudomonas￥fluoresc
ensSCB-10705) strain (Feikoken Bacterial Serial No. 9138)
), Pseudomonas florescens SCB-108
07(￥Pseudomonas￥fluoresc
ensSCB-10807) strain (Feikoken Bacterial Serial No. 9139)
), Xanthomonas sp. SCB-12801 (¥X
anthomonas￥ sp. SCB-12801 strain (Feikoken Bacterial Serial No. 9140) and Pseudomonas sp.
．． SCB-16701 (￥Pseudomonas￥
sp. SCB-16701) strain (Feikoken Bacillus No. 9141)
By attaching and growing Pythium bacteria and Rhizoctonia bacteria on vegetables, soil diseases of vegetables caused by Pythium bacteria and Rhizoctonia bacteria can be suppressed.
How to grow healthy vegetables (2) The method according to claim 1, characterized in that it suppresses seedling damping-off of cucurbitaceous vegetables. (3) Claim 1, characterized in that the method for adhering to and growing vegetables involves coating vegetable seeds.
Method described in section (4) The method according to claim 1, wherein the method for adhering to and growing vegetables includes immersing vegetable seedlings in a bacterial culture solution. (5) Xanthomonas sp. SCB-12801 (￥
Xanthomonas sp. SCB-12801
) strain (Feikoken Bacillus No. 9140) (6) Pseudomonas sp. SCB-16701 (￥
Pseudomonas sp. SCB-16701
) strain (Feikoken Bacillus No. 9141)